CPEB2-activated axonal translation of VGLUT2 mRNA promotes glutamatergic transmission and presynaptic plasticity

Background Local translation at synapses is important for rapidly remodeling the synaptic proteome to sustain long-term plasticity and memory. While the regulatory mechanisms underlying memory-associated local translation have been widely elucidated in the postsynaptic/dendritic region, there is no direct evidence for which RNA-binding protein (RBP) in axons controls target-specific mRNA translation to promote long-term potentiation (LTP) and memory. We previously reported that translation controlled by cytoplasmic polyadenylation element binding protein 2 (CPEB2) is important for postsynaptic plasticity and memory. Here, we investigated whether CPEB2 regulates axonal translation to support presynaptic plasticity. Methods Behavioral and electrophysiological assessments were conducted in mice with pan neuron/glia- or glutamatergic neuron-specific knockout of CPEB2. Hippocampal Schaffer collateral (SC)-CA1 and temporoammonic (TA)-CA1 pathways were electro-recorded to monitor synaptic transmission and LTP evoked by 4 trains of high-frequency stimulation. RNA immunoprecipitation, coupled with bioinformatics analysis, were used to unveil CPEB2-binding axonal RNA candidates associated with learning, which were further validated by Western blotting and luciferase reporter assays. Adeno-associated viruses expressing Cre recombinase were stereotaxically delivered to the pre- or post-synaptic region of the TA circuit to ablate Cpeb2 for further electrophysiological investigation. Biochemically isolated synaptosomes and axotomized neurons cultured on a microfluidic platform were applied to measure axonal protein synthesis and FM4-64FX-loaded synaptic vesicles. Results Electrophysiological analysis of hippocampal CA1 neurons detected abnormal excitability and vesicle release probability in CPEB2-depleted SC and TA afferents, so we cross-compared the CPEB2-immunoprecipitated transcriptome with a learning-induced axonal translatome in the adult cortex to identify axonal targets possibly regulated by CPEB2. We validated that Slc17a6, encoding vesicular glutamate transporter 2 (VGLUT2), is translationally upregulated by CPEB2. Conditional knockout of CPEB2 in VGLUT2-expressing glutamatergic neurons impaired consolidation of hippocampus-dependent memory in mice. Presynaptic-specific ablation of Cpeb2 in VGLUT2-dominated TA afferents was sufficient to attenuate protein synthesis-dependent LTP. Moreover, blocking activity-induced axonal Slc17a6 translation by CPEB2 deficiency or cycloheximide diminished the releasable pool of VGLUT2-containing synaptic vesicles. Conclusions We identified 272 CPEB2-binding transcripts with altered axonal translation post-learning and established a causal link between CPEB2-driven axonal synthesis of VGLUT2 and presynaptic translation-dependent LTP. These findings extend our understanding of memory-related translational control mechanisms in the presynaptic compartment. Supplementary Information The online version contains supplementary material available at 10.1186/s12929-024-01061-2.


Behavioral Assays
All assays were conducted during 13:30-17:30 light phase by observers who did not know the genotype of the mice until the tests had been completed.Behavioral tasks were performed with 2-to 4-month-old male littermates following the previous protocols with modifications (1)(2)(3).
Open field: Each mouse was released into a corner of the arena and allowed to explore for 10 min.The recorded moving trace of each mouse was analyzed by using the TopScan system (CleverSys).
Elevated plus maze: The elevated plus maze (EPM) consisted of two open arms with 1 cm ledges and two enclosed arms with 15 cm walls.The maze was elevated to a height of 50 cm above the floor during the task.The mouse behaviors were recorded in a 5 min testing period and analyzed by using the TopScan system (CleverSys).
Morris water maze: mice were trained for the hidden platform version during acquisition, which consisted of 4 trials daily for 4 consecutive days.Mice were released to the MWM from all other 3 quadrants except the target quadrant and were allowed to stand on the platform for 15 sec before being transferred back to cages.The probe trial was performed on the 5 th day in the water maze without a platform.For the visible MWM task, the escape platform marked by a flag was placed to measure the swimming ability and visual acuity of the mice.The maximal swimming duration for all trials was 1 min with a 15-min inter-trial interval.The trajectories of the mice were recorded and analyzed by using TrackMot (Singa Technology Corp.Corp., Taiwan).A CPEB2-cKO Nes and B CPEB2-cKO Vglut2 male mice (3 to 5 months old) were used for LTP recording induced by 1X HFS in the TA-CA1 circuit.C The same recording was performed in CPEB2-cWT male mice (4 to 5 months old) injected with AAV8-GFP (4.2 x 10 9 vg) or AAV9-Cre (1.62 x 10 10 vg) at the lateral entorhinal cortex (@EC).Pre-synaptic deletion of CPEB2 had no effect on 1X HFS-evoked LTP in the TA-CA1 circuit.Numbers in parentheses (n/N) represent the number of recorded slices (n) and mice (N).Sample traces with vertical scales of 0.5 mV and horizontal scales of 10 ms presented in the same manner as described in Fig. 1.Data are mean ± SEM.DIV20 rat cortical neurons were treated with 20 μg/ml cycloheximide (CHX) to block new protein synthesis for the indicated times and then harvested for immunoblotting of denoted proteins.The amount of individual protein was expressed as a relative percentage to the time zero which was arbitrarily set to 100.Data are mean ± SEM from 3 independent cultures.*P < 0.05, **P < 0.01 and ***P < 0.001, two-way ANOVA.
Fig. S1 Impaired spatial learning and memory in CPEB2-cKO Nes mice.Adult male mice at 2 to 4 months old were used for behavior assays.A Open-field. Representative moving traces in the open arena during the first 10 min and the quantified entry times, duration, moving distance and velocity.B Elevated plus maze (EPM).Representative 5-min moving traces in the EPM and the quantified entry times, duration and moving distance in the open versus closed arms.C Morris water maze (MWM).Mice were trained with 4 trials per day for 4 consecutive days, and the escape latency to the platform was averaged from 4 trials.D The MWM probe test on day 5 recorded the percentage of time (total 60 sec) spent in each quadrant.E In the visual MWM task, the velocity and latency reflected the swimming ability and visual acuity, respectively.Data are mean ± SEM. *P < 0.05 and **P < 0.01, Student's t test and two-way ANOVA with Fisher's LSD post-hoc test.The number of mice for behavioral assays is in parentheses.
Fig. S4No evident Cre-mediated effect on electrophysiological properties in the TA-CA1 pathway.A AAV9-GFP (1.62 x 10 10 vg), AAV9-Cre (1.62 x 10 10 vg) or AAV8-GFP (4.2 x 10 9 vg) were intracranially injected to the lateral entorhinal cortex (@EC, marked in green dots) of 5month-old wild-type (WT) male mice.Approximately 3 weeks after intracranial delivery of AAV, hippocampal slices were used for field recording.B The input-output responses and C paired-pulse facilitation in the TA-CA1 pathway.D The 4X HFS-evoked LTP in WT mice injected with denoted viruses was compared to that in CPEB2-cWT mice injected with AAV9-Cre (data from Fig.5D).Numbers in parentheses (n/N) represent the number of recorded slices (n) and mice (N).Sample traces were presented in the same manner as described in Fig 1.Data are mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001, compared to the cWT+AAV9-Cre group, two-way ANOVA with Fisher's LSD post hoc test.

Table S1 Antibodies and dyes used in the study.
The catalogue number and dilution information of antibodies and dyes.